N-(butoxymethyl)acrylamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro gene mutation study in bacteria was conducted on the read-across substance, acrylamide, using methodology equivalent to OECD test guideline 471. The read-across substance was negative for genotoxicity.

 

An in vitro mammalian cell gene mutation assay was conducted with the read-across substance, acrylamide, using methodology equivalent to OECD test Guideline 476. The read-across substance was negative for genotoxicity.

 

An in vitro mammalian chromosome aberration test was conducted with the read-across substance, acrylamide, according to OECD test Guideline 473. The assay was positive for clastogenicity under the conditions of the test as the read-across substance induced chromosomal structural changes and polyploidy.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See the Analogue Approach Report attached in Section 13 of the IUCLID dossier.

Reason / purpose for cross-reference:

other: Read across to target substance

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine biosynthetic pathway

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix from Aroclor 1254-induced Wistar rats

Test concentrations with justification for top dose:

0,1, 2, 5, 10, 20, 50, 70, 100 mg/plate

Vehicle / solvent:

Vehicle/solvent used: DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

0.2 and 1 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: no
- Exposure duration: 5 days
NUMBER OF REPLICATIONS: 3
NUMBER OF CELLS EVALUATED: 10E9 cells per ml
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Number of revertants per plate

Statistics:

Student's t-test

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At highest dose level

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At highest dose level

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At highest concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At the highest dose level

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At the highest dose level

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative

Acrylamide did not increase the number of reverse mutations in the Ames test at concentrations up to 100 mg/plate either with or without enzymatic activation. Results were consistently negative using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535, and TA 1537 in the presence and absence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See the Analogue Approach Report attached in Section 13 of the IUCLID dossier.

Reason / purpose for cross-reference:

other: Read across to target substance

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

0, 50, 150, 500, 1000, 1500 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: substance is totally soluble and stable in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Ethylmethanesulphonate and N-dimethylnitrosamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 16-24 hours
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
SELECTION AGENT (mutation assays): 6-thioguanine
NUMBER OF REPLICATIONS: 5
NUMBER OF CELLS EVALUATED: 2x10E5
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The total number of mutant colonies (per 10E6 surviving cells) observed at each concentration of test substance was compared to the total number of mutant colonies (per 10E6 surviving cells) observed in the negative control. A test substance showing a dose-dependent increase of mutation induction with at least one dose exhibiting a mutation frequency that was greater than or equal to 50X10E-6 per cell was considered a suspect mutagenic response

Statistics:

None

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative

The results for test article, Acrylamide, were negative in the CHO/HGPRT Mamalian Cell Forward Gene Mutation Assay according to criteria of the test protocol.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See the Analogue Approach Report attached in Section 13 of the IUCLID dossier.

Reason / purpose for cross-reference:

other: Read across to target substance

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable (chromosome assay)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Metabolic activation system:

none

Test concentrations with justification for top dose:

0, 0.5, 1, 2, 3, 4 and 5 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: test substance is totally soluble and stable in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

Methylnitrosonitroguanadine (MNNG)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period:
- Exposure duration: 24 hours
- Expression time (cells in growth medium): 20 and 40 hours
- Fixation time (start of exposure up to fixation or harvest of cells): according to Rothfels and Simonvitch, 1958
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): according to Rothfels and Simonvitch, 1958
NUMBER OF REPLICATIONS: 1
NUMBER OF CELLS EVALUATED: metaphase cells = 100, polypoidy = 500
DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency
OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

Cells with more than 33 chromosomes were counted as polyploid.

Statistics:

None

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

other: Clastogenicity: positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
positive

Acrylamide induces chromosomal structural changes and polyploidy.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo mammalian germ cell study (cytogenicity/chromosome aberration) of the read-across substance, acrylamide, was conducted using methodology equivalent to OECD Test Guideline 478 using a dominant lethal assay in rats. The results were positive.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See the Analogue Approach Report attached in Section 13 of the IUCLID dossier.

Reason / purpose for cross-reference:

other: Read across to target substance

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

rodent dominant lethal assay

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI.
- Age at study initiation: males: 11 weeks, females: 9 weeks at the start of the mating period
- Weight at study initiation: males: 306.8 to 383.6 g, females: 191.5 to 250.9 g
- Assigned to test groups randomly: yes, under following basis: One hundred fifty (150) males were assigned to six (6) groups, 25 males per group, by a stratified randomisation procedure to provide uniform body weights across groups at the initiation of the study. One hundred fifty (150) females, in the appropriate stage of estrus, were also assigned to the six (6) groups, 25 females per group, by a stratified randomization procedure to provide uniform body weights across groups at the initiation of mating.
- Fasting period before study: yes
- Housing: solid bottom polycarbonate cages with stainless steel wire lids, with Sani-Chips animal bedding
- Diet: ad libitum No. 5002 Purina Certified Rodent Chow
- Water: ad libitum tap water
- Acclimation period: 4 to 6-day quarantine period for the males and the 0 to 3 day quarantine period for the females
ENVIRONMENTAL CONDITIONS
- Temperature: 70.8 - 73.8°F.
- Humidity (%):46.4 - 64.5%.
- Photoperiod : 12:12 hour Iight/dark cycle
IN-LIFE DATES: From: May 7, 1997 To: June 7, 1997

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: water
- Justification for choice of solvent/vehicle: test article is totally water-soluble
- Purity: sterile and distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Acrylamide monomer in vehicle or vehicle alone was administered by gavage, at a volume of 5.0 ml/kg, based on each animal's most recent body weight, to each male once daily for five consecutive days. The dosing solutions were administered in a syringe of appropriate volume attached to a 16 gauge, 2 inch curved dosing needle (Perfektum, Popper and Sons, New Hyde Park, NY). Gavage was chosen by the Sponsor as the route of administration to duplicate the route employed by Sublet et al. (1989).

Duration of treatment / exposure:

5 days

Frequency of treatment:

Daily

Post exposure period:

8 days prior to mating

Remarks:

Doses / Concentrations:
0, 5, 15, 30, 45 and 60 mg/kg/day
Basis:
nominal in water
equivalent to 0.0, 1.0, 3.0, 6.0, 9.0 and 12.0 mg/ml at a dosing volume of 5.0 ml/kg

No. of animals per sex per dose:

25 males and 25 females (females were not dosed)

Control animals:

yes, concurrent vehicle

Positive control(s):

No

Tissues and cell types examined:

All study males in all groups were weighed and subjected to a complete gross necropsy. The gross necropsy included examination of the external surfaces; all orifices; cranial cavity; carcass; external and cut surfaces of the brain and spinal cord; the thoracic, abdominal, and pelvic cavities and their viscera; and cervical tissues and organs. Sperm motility (motile and progressively motile) and sperm beat cross frequency were assessed immediately after necropsy; number of sperm (per sample and per g epididymis) was evaluated at a later date using appropriately retained sperm samples. For the males not perfused in vivo (20 per group), one cauda epididymis was immediately removed, weighed and seminal fluid from the cauda was assessed for sperm number and motility.
Mated females were weighed and sacrificed at mid-pregnancy, on gd 15. The gravid uterus with attached oviducts and one ovary was dissected free and removed. The total numbers of ovarian corpora lutea were counted. Total implantation sites RTI-65C-6B21-200 were also counted and recorded. The status of each implantation site was also documented: resorption (early, late and/or total) or live implantation. If any uterus appeared non-gravid upon initial inspection, it was placed in 10% ammonium sulphide for confirmation of pregnancy status. If implantation sites appeared after staining, the total number in the uterus was recorded. Each female and all her tissues were discarded. If a female was not pregnant at scheduled sacrifice, her pregnancy status was included in the calculation of the various reproductive indices, but her data were not entered or summarized since these data were reported only for pregnant females.

Details of tissue and slide preparation:

Gross lesions were retained in fixative. Five (5) males per group were preserved by in vivo perfusion fixation with glutaraldehyde prior to necropsy. The testes and one sciatic nerve of all study males (both perfused and non-perfused) were retained in fixative for possible subsequent histopathology. The other epididymis and both testes per non-perfused male were retained in fixative for possible subsequent histopathologic examination. The sciatic nerves from the perfused males, five/group, were embedded in paraffn, sectioned at approximately 3-4 microns, stained with hematoxylin and eosin and examined histopathologically. Subsequently. additional sections of sciatic nerves from these perfused males were cut, stained with Holm's Silver/Luxol Fast Blue Stain (which gives comparable results as Bodian's Fast Blue Stain) to better differentiate the axon and myelin sheath, and examined histopathologically (second report, Appendix III). The testes and sciatic nerves from non-perfused males were not processed or examined; the testes from the perfused males were also not processed or examined histopathologically.

Evaluation criteria:

Males:
Mating index (%) = (No. impregnating females / No. males paired) x 100
Fertility index (%) = (No. males siring litters / No. impregnating females) x 100
Pregnancy index = (No. pregnant females / No. males impregnating females) x 100
Females:
Preimplantation loss (%), = ((No. corpora lutea – No.implantations) / No.corpora lutea) x 100
Postimplantation loss = ((No. total implantations - No. live implantations) / No. total implantations) x 100
Frequency of dominant lethal factors, FL %, = (1- (No. live implantations per female of test group / No. live implantations per female of control group)) x 100

Statistics:

Quantitative continuous data were compared among treatment groups against vehicle control group using Bartlett's test for homogeneity of variances. If this indicated lack of homogeneity of variances (p<0.001), nonparametric statistical tests were employed for continuous variables. If Bartlett's test indicated homogeneous variances (p>0.001), parametric statistical tests were employed for continuous variables. Parametric statistical procedures were applied to selected measures from this study. Appropriate GLM for proposed Analyses of Variance was used to determine significance of dosage-response relationship (Test for Linear Trend) and determine if significant dosage effects had occurred for selected measures. When a significant (p<0.05) main effect for dosage occurred, Dunnett's Multiple Comparison was used to compare each dosed group to the control group for that measure. A 1- or 2-tailed test was used for all pairwise comparisons to vehicle control group. Nonparametric tests included Kruskal-Wallis Test to determine if significant differences were present among groups, followed by Mann-Whitney U test for pairwise comparisons to designated control group, if Kruskal. Wallis test was significant. Jonckheere's test for k independent samples was used to identify significant dose-response trends for nonparametric continuous data. Frequency data were analyzed by Chi-Square Test for Independence for differences among treatment groups, and by Cochran-Armitage Test for Linear Trend on Proportions. When Chi-Square revealed significant (p<0.05) differences among groups, a 2-tailed Fisher's Exact Probability Test, with appropriate adjustments for multiple comparisons, was used for pairwise comparisons between each dosed and control group. For all statistical tests, the significance limit of 0.05 (1- or 2-tailed) was used as criterion for statistical significance. A test for statistical outliers was performed on body weights.

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Remarks:

at 15, 30, 45 and 60 mg/kg/day

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

This study was performed to duplicate, to the extent possible, a portion of the study design of Sublet et al. (1989). Their design involved male Long-Evans rats dosed with acrylamide monomer at 0, 5, 15, 30. 45 or 60 mg/kg/day for five days (sd 1-5), then serially mated to naive females for 10 weeks beginning on sd 8. Effects in the Sublet study included reduced fertility and increased pre- and post-implantation loss at 15 through 60 mg/kg/day, primarily over the first three weeks post-treatment. The effects on week 1 appeared to result from an interference with sperm transport since there were reductions in percent females with uterine sperm (but not vaginal sperm) at 15 mg/kg/day (not significant) and 45 mg/kg/day (statistically significant), following a single ejaculation by treated males. Effects on sperm motility and curvilinear velocity were present at 45 mg/kg/day on week 3; there were no effects of treatment on sperm count, sperm linearity or straight line velocity (Sublet et al., 1989). There were no reported parameters of general toxicity or neurotoxicity for the males. Male systemic toxicity was present at 15, 30, 45 and 60 mg/kg/day, based on body weight changes during the dosing period (sd 1-5) and for the entire evaluation period until mating (sd 1-8). Grip strength in the hind limbs (but not the forelimbs) was significantly reduced at 60 mg/kg/day and treatment-related clinical observations were present at 45 and 60 mg/kg/day. There was no evidence of the characteristic acrylamide-induced peripheral neuropathy in the sciatic nerves from perfused males, e.g., no evidence for axonal swelling or degeneration after hematoxylin and eosin staining or after Holm's Silver/Luxol Fast Blue Stain which is a better stain to identify these effects (e.g., Friedman et al., 1997). Male reproductive toxicity was clearly present at 45 and 60 mg/kg/day. The effects included reduced mating, fertility and pregnancy indices (no values statistically significantly different from control males), increased post-implantation loss/litter (statistically significantly different at 45 and 60 mg/kg/day), decreased live implants per litter at 45 and 60 mg/kg/day (none statistically significantly different), reduced percent progressively motile sperm (possibly at 60 mg/kg/day, not statistically significantly different), and increased beat cross frequency (possibly but not significantly increased at 45 mg/kg/day, significantly increased at 60 mg/kg/day). Each of these parameters was affected by acrylamide monomer at doses as low as 15 mg/kg/day, with no statistically significant pairwise differences to the concurrent control group values. Reproductive parameters that were not affected included pre-implantation loss/litter and percent motile sperm. Epididymal sperm concentration was increased, but not statistically significantly, in all acrylamide-dosed groups with the highest value at 60 mg/kg/day (Text Table B). The increased sperm concentration in the cauda epididymis (site of sampling) may be due to reduced ejaculatory behavior or to interference with sperm transport to the vas deferens in acrylamide-treated males. The effects of acrylamide monomer on mating and fertility do not change; effects are obvious at 60 mg/kg/day and apparent at 15-45 mg/kg/day. The overall mating performance of the males is improved, confirming that the females used on the first pairing day were probably in early proestrus when paired and did not cycle to estrus during the overnight cohabitation period. The values for fertility index did not change for all groups except the 5 mg/kg/day group in which one female became sperm positive (but was not pregnant) on the first mating day.

Conclusions:

Interpretation of results (migrated information): positive
Systemic and reproductive toxicity was observed at 15, 30, 45 and 60 mg/kg/day. Statistically significant indicators of systemic toxicity occurred at 15-60 mg/kg/day, and statistically significant indicators of reproductive toxicity occurred at 45-60 mg/kg/day (with effects present at 15-30 mg/kg/day with no statistically significant differences). Therefore, the No Observed Adverse Effect Level (NOAEL) for both systemic and reproductive toxicity was 5 mg/kg/day in Long-Evans male rats under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Mode of Action Analysis / Human Relevance Framework

Studies using bacterial mutagenesis assays have consistently failed to show a mutagenic response for acrylamide. Data from mammalian cell mutagenesis assays have been conflicting, but generally do not provide evidence of mutagenicity of acrylamide. Positive results have been reported in the mouse lymphoma L5178Y cell mutagenesis assay, but the response, consisting almost exclusively of small-colony mutants, is indicative of chromosomal effects rather than being true point mutations. Studies using assays that measure direct damage to DNA have also given negative results.

In vitro tests for induction of sister chromatid exchanges (SCE) have been conflicting, with one lab reporting a positive response with acrylamide and a second one reporting a negative result. Two laboratories have evaluated acrylamide in the rat hepatocyte DNA repair assay. One laboratory reported a positive response for acrylamide, but there were numerous technical problems with the conduct and interpretation of these results. A second study conducted in a well-recognized laboratory reported negative results in this assay.

There have been numerous reports on the cytogenetic effects of acrylamide on bone marrow, spermatogonia, and other cell types. Although some reports are conflicting, the general consensus is that acrylamide is a potent clastogen in bone marrow, but results in spermatogonia are equivocal. Results in other test systems likewise produce conflicting results. Two laboratories reported negative results in cell transformation assays using BALB/3T3 or C3H/10T1/2 cells; a third laboratory reported positive cell transformation results in C3H/10T1/2 and NIH/3T3 cells. Two laboratories reported negative results in the sex-linked recessive lethal assay in Drosophila melanogaster, but one of these laboratories reported a positive result in the somatic mutation and recombination test (SMART) in Drosophila.

The experimental data suggests a mode of action of acrylamide that does not involve binding to DNA. Studies to assess covalent binding of acrylamide to macromolecules have consistently failed to demonstrate significant binding to DNA. In contrast, binding to microtubule proteins is extensive, which could account for many of the chromosomal effects of acrylamide. A study that evaluated binding of acrylamide to spermatogonial cells in mice revealed that significant binding was observed in late-spermatid to early spermatozoa stages, the same stages that are most sensitive to the action of acrylamide. Evaluation of the separate fractions revealed that very little acrylamide is bound to DNA, and the binding of acrylamide to protamine accounts for virtually all of the binding.

Additional information

The genotoxic potential of the read-across substance, acrylamide, was assessed in an in vitro gene mutation study in bacteria using methodology equivalent to the OECD test guideline 471 using a bacterial reverse mutation assay with and without metabolic activation. The read-across substance was added to the agar at 0, 1, 2, 5, 10, 20, 50, 70 and 100 mg/plate for Salmonella typhimurium strains TA 1525, TA 1537, TA 98, TA 100 and TA 102 for 5 days. Cytotoxicity was observed at the highest dose level in all strains but there was no increase in the number of reverse mutations in the Ames test either with or without metabolic activation and therefore the read-across substance was negative for genotoxicity.

 

An in vitro mammalian cell gene mutation assay was conducted with the read-across substance, acrylamide, using methodology equivalent to OECD test Guideline 476 using Chinese hamster ovary cells with and without metabolic activation. The read-across substance was added to the agar at 0, 50, 150, 500, 1000 and 1500 µg/mL. Cytotoxicity was observed but there was no indication of genotoxicity with or without metabolic activation and therefore the read-across substance was negative for genotoxicity.

 

An in vitro mammalian chromosome aberration test was conducted with the read-across substance using methodology equivalent to OECD test Guideline 473using Chinese hamster lung fibroblasts without metabolic activation. The read-across substance was added to the medium at 0, 0.5, 1, 2, 3, 4 and 5 mM. Cells were exposed for 24 hours followed by 20 and 40 hours of expression time. Cells with more than 33 chromosomes were counted as polyploid. The assay was positive for clastogenicity under the conditions of the test as the read-across substance induced chromosomal structural changes and polyploidy.

 

An in vivo mammalian germ cell study (cytogenicity/chromosome aberration) of the read-across substance, acrylamide, was conducted using methodology equivalent to OECD Test Guideline 478 using a dominant lethal assay in rats. The read-across substance was administered to male and female rats by oral gavage daily for 5 days at 0, 5, 15, 30, 45 and 60 mg/kg/day.

The purpose of the dominant lethal test is to investigate whether acrylamide is a primary reproductive toxicant producing mutations resulting from chromosomal aberrations in germ cells, or whether the effects on male reproduction were due to its systemic toxicity.

Dominant lethal mutations cause embryonic or foetal death. Induction of dominant lethal mutations after exposure to a test material indicates that the chemical has affected the germ cells of the animal. The dominant lethality of a test chemical is determined by comparing the live implants per female in the treated group with the live implants per female in the vehicle / solvent control group.

The post-implantation loss is then calculated by determining the ratio of dead to total implants from the treated group compared to the ratio of dead to total implants from the vehicle / solvent control group. 

Pre-implantation loss is calculated as the difference between the number of copora lutea and the number of implants or as a reduction in the average number of implants per female in comparison with the control matings.

The dominant lethal study was performed in duplicate in male Long-Evans rats dosed with the acrylamide monomer at 0, 5, 15, 30, 45 or 60 mg/kg/day for five days, then serially mated to naïve females for 10 weeks beginning on sd 8. Effects in the sub-let study included reduced fertility and increased pre-and post-implantation loss from 15 mg/kg/day and above, primarily over the first three weeks post-treatment. Male reproductive toxicity was clearly present and increased post-implantation loss/litter was statistically significant at 35 and 60 mg/kg/day. Pre-implantation loss/litter was not affected.

The statistically significant difference in post-implantation loss indicates that the read-across material, acrylamide is positive for genetic toxicity in vivo.

Justification for classification or non-classification

Acrylamide has a harmonised classification for mutagenicity as Category 1B.